PL222208B1 - Camptothecin derivatives having antitumor activity - Google Patents

Abstract

Derivatives of camptothecin of formula (I) <CHEM> wherein the groups R1, R2 and R3 are as defined in the description are disclosed. The compounds of formula (I) are endowed with antitumor activity and show a good therapeutic index. Processes for the preparation of the compounds of formula (I) and their use in the preparation of medicaments useful in the treatment of tumors are also disclosed.

Description

The present invention relates to compounds with anti-tumor activity, in particular to novel camptothecin derivatives, methods for their preparation, their use as anti-tumor drugs and pharmaceutical compositions containing these compounds as active ingredients.

Camptothecin is an alkaloid that was isolated by Wall et al. (J. Am. Chem. Soc.

88, 3888-3890 (1966)) for the first time from a tree Camptoteca acuminata, native to China, family Nyssaceae.

Its molecule contains a pentacyclic structure having an E-ring lactone which is essential for cytotoxicity.

The drug has a broad spectrum of anti-cancer activity, especially against colon cancer, other solid leukemia tumors, and the first clinical trials were carried out in the early 1970s. Camptothecin (hereinafter CPT) has low water solubility and the National Cancer Institute (NCI) has prepared the sodium salt (NSC 100880) which is soluble in water. Phase I and II clinical trials were not completed due to the high toxicity of the compound (hemorrhagic cystitis, gastrointestinal toxicity such as nausea, vomiting, diarrhea, and bone marrow suppression, especially leukopenia and thrombo-cytopenia. In each case, salt. sodium shows lower activity than CPT because, at pH 7.4, the inactive form (open ring) dominates over the active lactone form (closed ring) which is dominant at pH <4.0.

Subsequently, many CFT analogs were synthesized to obtain compounds with lower toxicity and higher water solubility.

Two drugs are labeled, Irinotecan (CPT-11), trademarked Camptosar® by Upjohn and Topotecan, trademarked Hymcamptamin® or Thycantin®, trademarked by Smith Kline & Beecham. Other derivatives are at various stages of clinical development in Phase II such as NSC-603071 (9-amino-camptothecin), 9-NC or 9-nitrocamptothecin, oral prodrugs converted to 9-aminocamptothecin, GG-211 (GI 147211), and DX-8591f, the latter water-soluble. All derivatives identified so far contain a five-ring parent structure essential for their toxicity. Modifications to the first ring, such as in the drugs mentioned above, have been shown to increase the solubility and allow a higher drug tolerance.

Water-soluble Irinotecan has been approved for the treatment of many solid tumors and ascites (colon-rectum, skin, stomach, breast, small and non-small cell lung, cervical and ovarian cancer, and non-Hodgkin's lymphoma). In addition, Irinotecan is active in Topotecan, vincristine or melphaIan resistant solid tumors and marginal drug resistant MDR-1 cells. The active metabolite was identified as a 10-hydroxy derivative (SN-38) produced by the action of carboxylesterases. CPT-11 showed good activity when administered by various routes, such as parenteral, intravenous, orally (Costin D., Potmhexyl M. Advances in Farmacol. 29B, 51-72 1994).

CPT-11 was also administered with cisplatin or ethoposide, achieving a synergistic effect due to its ability to interfere with DNA repair. Also in this case, leukopenia grades 3 and 4 and diarrhea appeared (Sinha B. K. (1995) Topoisomerase inhibitors. Drugs 49, 11-19, 1995).

Topotecan has significant oral bioavailability. Oral administration has shown to be convenient to obtain sustained drug exposure without the use of the necessary temporary catheters (Rothenberg M.L. Annals of Oncology 8, 837-855, 1997). Also, this water-soluble CPT analog shows activity against various types of tumors with different routes of administration, parenterally, intravenously, subcutaneously, orally. More promising effects were obtained by intravenous infusion of Topotecan hydrochloride for 5 days, in various neoplasms such as small and non-small cell lung, ovary, breast, stomach, liver, prostate, soft tissue sarcoma, head and neck, esophagus, resistant colon-rectum , glioblastoma multiforme, chronic and acute myelocytic leukemia. Again, several side effects such as neutropenia and thrombocytopenia were observed, while gastrointestinal toxicity such as nausea, vomiting and diarrhea were milder.

The major transformation and elimination of drug pathways has been proven to involve lactone hydrolysis and urinary excretion: in fact, 50% of the lactone form is hydrolyzed by ring opening 30 minutes after infusion. Topotecan crosses the haematoencephalic barrier 10 minutes after infusion (30% in liquid

In relation to plasma). In contrast, camptothecin does not cross the hematoencephalic barrier in significant amounts, possibly due to protein binding.

The clinical development of 9-aminocamptothecin was hampered by its lack of water solubility. Recently, a colloidal dispersion has been produced which allows it to enter Phase II clinical trials. Prolonged exposure (from 72 hours to 21 days) appears to be important for demonstrating anti-tumor activity because of its short half-life (Dahut et al., 1994). There have been reports of reactions in patients suffering from untreated colorectal and breast cancer and refractory lymphoma. The activity demonstrated against Pgp-positive tumors suggests no cross-resistance against resistant MDR-1 cells. Again, bone marrow and gastrointestinal toxicity has been observed.

Lurtotecan is the most soluble analogue with in vitro activity comparable to Topotecan. Two regimes were adopted: one 30 min. Daily infusion for 5 days every 3 weeks and one 72-hour infusion once every 3 weeks. The reactions of patients suffering from cancers of the cervix, ovary, breast and liver have been observed. Also in this case hematical toxicity was detected.

The formula is as follows:

9-Nitrocamptothecin is an orally administered prodrug that is rapidly converted to 9-aminocamptothecin upon administration. Reactions have been observed in patients suffering from pancreatic, ovarian and breast cancer.

However, the major part of tumor cells is very sensitive to topoisomerase I inhibitors, due to the high levels of the enzyme, certain tumor lines appear to be resistant. This is due to mechanisms other than overexpression of the MDR 1 and multidrug resistance associated protein (MRP) genes and their products, P-glycoproteins (Pgp) and MRP proteins, respectively, for which

Topotecan or CPT-11 are not good substrates (Kawato Yet al J. Farm. Farmacol. 45, 444-448, (1993)).

In fact, it has been observed that some resistant tumor cells contain the topo I mutant forms, respectively, the formation of the topo I-DNA complex is damaged, or some cells lack the carboxylesterase activity necessary to convert CPT-11 into the active metabolite SN-38 and are therefore resistant to this drug. (Rothenberg, 1997, ibid.).

Among the drugs used in cancer therapy, the interest in inhibitors of topoisomerase I enzymes has been attributed to the following factors: a) efficacy against tumors inherently resistant to conventional drugs, including topoisomerase II inhibitors; b) the levels of the topo I enzyme remain elevated in all phases of the cycle; c) many tumors present high levels of the target enzyme; d) lack of recognition by proteins due to the phenomenon of multi-drug resistance (Pgp or MRP) and the absence of detoxification enzyme metabolism associated with the glutathione-dependent system (glutathione peroxidase and glutathione S-transferase) (Gerrits CJH., et al., Brit. J Cancer 76,952-962).

Due to the potential clinical advantages of topoisomerase inhibitors, both in terms of antitumor activity, studied in a number of cancers, and in inducing pharmacological resistance, the current research is aimed at identifying topo I inhibitors with low toxicity in relation to those demonstrated by the drugs on the market. or in the clinical phase. Indicators of the relative potency of camptothecin analogues include a) proper activity

Inhibiting topoisomerase I; b) the average duration of the drugs; c) interaction with plasma proteins; d) the ratio between the circulating active form (Iactone) and the inactive form (carboxylate); e) drug sensitivity with respect to P-glycoprotein or MRP mediated cell efflux; f) stability of binding to topoisomerase I (Rothenberg, 1997, ibid.).

Among the more serious adverse effects of Irinotecan and other camptothecin derivatives, bone marrow atrophy and gastrointestinal toxicity such as diarrhea and vomiting have been observed. Diarrhea may be early or late onset and may be a dose limiting factor. Vomiting and subsequent diarrhea are caused by many anti-cancer drugs, while the early diarrhea during or immediately after infusion is almost specific to Irinotecan and some camptothecin derivatives.

The toxic effects occur mainly in the intestinal tract.

To reduce diarrhea, CPT-11 has been administered in some clinical trials in combination with loperamide, a synthetic opioid, an agonist of intestinal mu-opioid receptors (Abigerges, 1994; Abigerges, 1995), as well as an enkephalinase inhibitor (acetorphan) or with ondansetron. an antagonist of 5-HT3 receptors, or with difenidramine, an antagonist of H1 receptors.

To date, the problems associated with the use of camptothecin derivatives as anti-cancer drugs can be summarized in the following points:

- camptothecin (CPT), and many of its active derivatives have low water solubility;

the further derivatives have severe side effects for the gastrointestinal tract and the bone marrow;

- some tumor lines have become resistant to topoisomerase I inhibitors;

- there is a constant search for better therapeutic indicators.

WO 97/31003, incorporated herein by reference, discloses camptothecin derivatives substituted at positions 7, 9 and 10. Position 7 provides the following substitutions: -CN, -CH (CN) -R4, -CH = C (CN) -R4, -CH2-CH = C (CN) -R4, -C (= NOH) -NH2, -CH = C (NO2) -R4, -CH (CN) -R5, -CH (CH2NO2) -R5, 5-tetrazolyl , 2- (4,5-dihydroxazolyl), 1,2,4-oxadiazolidin-3-yl-5-one, wherein R4 is hydrogen, straight or branched alkyl having 1 to 6 carbon atoms, nitrile, carboxyalkoxy. Of these possible compounds, WO 97/31003 only discloses camptothecin derivatives having a -CN group and a -CH = C (CN) 2 group at position 7, with positions 9 and 10 unsubstituted.

Among these compounds, 7-nitrile (R4 = -CN), hereinafter CPT 83, with cytotoxic activity on non-small cell lung cancer (non-SCLC, H-460), appears to be the most preferred. This cell line is intrinsically resistant to cytotoxic therapy and is only moderately responsive to topoisomerase I inhibitors, despite overexpression of the target enzyme. CPT 83 is more reactive than Topotecan considered as a reference compound and in general it offers a better pharmacological profile, even in terms of tolerability, and thus a better therapeutic index.

CPT 83 is obtained by a synthesis involving oxidation of 7-hydroxymethyl camptothecin to camptothecin 7-aldehyde, transformation to oxime and finally conversion to nitrile.

The starting compound and intermediates are described by Sawada et al. Chem. Farm. Bull. 39, (10) 2574 (1991). This publication contains reference to the 1981 family of patents, for example, Patent Application EP 0056692, published in 1982, incorporated herein by reference. These publications disclose, among others, compounds of 7-aldehyde-camptothecin and its oxime. The utility of these derivatives lies in the preparation of compounds with low toxicity antitumor activity starting from 7-hydroxymethylcamptothecin. In a paper published in Chem. Farm. Bull. 39, (10) 2574 (1991), the authors prove that, with respect to camptothecin, the 7-alkyl and 7-acyloxymethyl derivatives not disclosed in the above-mentioned patent application are more active against the L1210 murine leukemia cell lines, and less active with respect to camptothecin, in compounds with 7-substituents of a highly polar nature, such as hydrazones and oximes -CH (= NOH).

It has been surprisingly found that camptothecin having an O-substituted alkyloxime at the 7-position are endowed with antitumor activity greater than that of the compound called Topotecan. More surprisingly, it has been found that camptothecins bearing the enamine group at position 7 are also endowed with anti-tumor activity. The above compounds have a better therapeutic index.

Accordingly, the present invention relates to the compounds of formula (I)

PL 222 208 B1

wherein:

R1 is the group -C (R5) = NO (n) R4 where R4 is straight or branched C1-C8-alkyl, or C6-C14-aryl selected from the group consisting of phenyl or benzyl, or heterocyclo-straight or branched C1- C8-alkyl wherein said heterocyclic group contains at least a heteroatom selected from the group consisting of a nitrogen atom optionally substituted with a (C1-C8) -alkyl group, and / or oxygen; wherein said heterocyclic group is selected from the group consisting of pyridyl, N-methylpiperidinyl, uracil, morpholinyl and oxiranyl; wherein the alkyl is optionally substituted with one or more groups selected from the group consisting of phenyl, -NR6R7 wherein R6 and R7 are hydrogen, or wherein said arylalkyl is optionally substituted with one or more groups selected from C1-alkyl, or halogen; or a pharmaceutically acceptable ester thereof; or

R4 is a glycosyl group selected from 6-D-glucosyl, optionally substituted with a ketal group selected from the group consisting of 1,3-dioxolyl;

n is 1;

R5 is H;

R2 is H;

R3 is hydrogen, hydroxy, alkoxy wherein alkoxy is methoxy; single isomers thereof, and pharmaceutically acceptable salts thereof.

The preferred compounds of the invention are:

7-methoxyiminomethylcamptothecin;

7-methoxyiminomethyl-10-hydroxycamptothecin;

7-t-butoxyiminomethylcamptothecin;

7-t-butoxyiminomethyl-10-hydroxycamptothecin;

7-t-butoxyiminomethyl-10-methoxycamptothecin;

7-triphenylmethoxyiminomethylcamptothecin;

7- (2-amino) ethoxyiminomethylcamptothecin;

7-benzyloxyiminomethylcamptothecin;

7-phenoxyiminomethylcamptothecin;

7-p-methylbenzyloxyiminomethylcamptothecin;

7-pentafluorobenzyloxyiminomethylcamptothecin;

7-p-phenylbenzyloxyiminomethylcamptothecin;

7-oxiranylmethoxyiminomethylcamptothecin;

7- [2- (1-uracil) ethoxy] iminomethylcamptothecin;

7- (4-pyridyl) methoxyiminomethylcamptothecin;

7 - [(N-methyl) -4-piperidinyl] methoxyiminomethyl camptothecin;

7 - [(2- (4-morpholininyl] ethoxy] iminomethylcamptothecin; and

7- (6-D-glucosyloxy) iminomethylcamptothecin.

The more preferred compounds of the invention are 7- (t-butoxy) iminomethylcamptothecin and 7-benzyloxy-iminomethyl camptothecin.

The invention also relates to a process for the preparation of compounds of formula (I) wherein R4 is as defined above, excluding aroyl, comprising reacting a compound of formula (Ia)

PL 222 208 B1

in which R1 is the group -C (R5) = O, where R5 is as defined in formula (I), R2 and R3 are as defined in formula (I), with a compound of formula (IIa) R ₄ O-NH ₂ , wherein R ₄ is as defined above to give compounds of formula (I), wherein R 1 is -C (R5) = N-OR 4, R 4 is as defined in formula (I), with the exception of aroyl.

In a preferred process according to the invention, the ratio between the compound of formula (Ia) and the compound of formula (IIa) is between 1: 3 and 3: 1.

The invention also includes a process for the preparation of compounds of formula (I) wherein R4 is as defined above, except aroyl, comprising reacting a compound of formula (Ia)

wherein R1 is a group

-C = N-OH, where R5 is as defined in formula (I), R2 and R3 are as defined in formula (I), with a halide of formula R4-X in which X is halogen and R4 is as defined above to give compounds of formula (I) in which R1 is a group of formula -C (R5) = N-OR4, R4 is as defined for formula (I), with the exception of aroyl.

The invention also relates to the above compounds as medicaments.

The invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention in admixture with pharmaceutically acceptable vehicles and excipients, wherein the therapeutically effective amount ranges from 2 mg / kg to 25 mg / kg.

The invention also encompasses the use of a compound as defined above for the preparation of a medicament useful in the treatment of tumors, preferably the compound is used for the manufacture of a medicament useful in the treatment of non-small cell lung cancer.

As examples of straight or branched C1-C8-alkyl groups within the scope of the present invention are methyl, ethyl, propyl, butyl, pentyl, octyl and their possible isomers, such as, for example, isopropyl, isobutyl, tert-butyl.

PL 222 208 B1

Examples of straight or branched C1-C8-alkylene groups are methylene, ethylidene, vinyl, allyl, propargyl, butylene, pentylene in which the carbon-carbon double bond, optionally in the presence of other carbon-carbon unsaturated bonds, may be located at different possible positions of the alkyl chain which may also be branched with acceptable isomers.

Examples of the C3-C10-cycloalkyl group are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, polycyclic groups such as for example adamantyl.

Examples of the C3-C10-cycloalkyl- (C1-C8)-straight or branched alkyl group are: cyclopropylmethyl, 2-cyclopropylethyl, 1-cyclopropylethyl, 3-cyclopropylpropyl, 2-cyclopropylpropyl, 1-cyclopropylpropyl, cyclobutylmethyl, 2-cyclobutyl, 2-cyclobutyl cyclobutylethyl, 3-cyclobutylpropyl, 2-cyclobutylpropyl, 1-cyclobutylpropyl, cyclohexylmethyl, 2-cyclohexylethyl, 1-cyclohexylethyl, 3-cyclohexylpropyl, 2-cyclohexylpropyl, 1-cyclohexylopropyl, 3-cyclohexylopropyl, 3-cyclohexylopropyl cyclohexylbutyl, 1-adamantylethyl, 2-adamantylethyl, adamantylmethyl.

Examples of (C6-C14) -aryl, or (C6-C14) -aryl- (C1-C8) -straight or branched alkyl group are, 1- or 2-naphthyl, anthryl, benzyl, 2-phenylethyl, 1-phenylethyl, 3-phenylpropyl, 2-anthrylopropyl, 1-anthrylpropyl, naphthylmethyl, 2-naphthylethyl, 1-naphthylethyl, 3-naphthylpropyl, 2-naphthylpropyl, 1-naphthylpropyl, cyclohexylmethyl, 5-phenylpentyl, 3-phenylpentyl 3-methylbutyl.

Examples of a heterocyclic or heterocyclo- (C1-C8) -linear or branched alkyl group are thienyl, quinolyl, pyridyl, N-methylethylpiperidinyl, 5-tetrazolyl, 2- (4,5-dihydroxazolyl), 1,2,4-oxadiazolidin-3 -yl-5-one, purine and pyrimidine bases, for example uracil, optionally substituted as shown in the above general definitions.

Examples of the (C1-C10) -aroyl group are benzoyl, naphthoyl.

Examples of the (C6-C10) arylsulfonyl group, optionally substituted with an alkyl group, are tosyl, benzenesulfonyl.

The halides are fluorine, chlorine, bromine, iodine.

Examples of the substituted group are pentafluorophenyl, 4-phenylbenzyl, 2,4-difluorobenzyl, 4-aminobutyl, 4-hydroxybutyl, dimethylaminoethyl, p-nitrobenzoyl, p-cyanobenzoyl.

An example of a polyaminoalkyl group is - (CH2) m-NR12- (CH2) p NR13- (CH2) q-NH2 where m, p are from 2 to 6 and q is from 0 to 6, including the extremes, R12 and R13 is a straight or branched (C1-C8) alkyl group, for example N- (4-aminobutyl) -2-aminoethyl, N- (3-aminopropyl) -4-aminobutyl, N- [N- (3-aminopropyl) - N- (4-aminobutyl)] - 3-aminopropyl.

Examples of a glycosyl group are 6-D-galactosyl, 6-D glucosyl, D-galactopyranosyl, where the glycosyl group is optionally protected with a suitable ketal group, for example isopropylidene.

Examples of pharmaceutically acceptable salts are, in the case of basic nitrogen atoms, salts with pharmaceutically acceptable acids, both inorganic and organic, such as, for example, hydrochloric, sulfuric, acetic acid, or, in the case of acidic groups such as carboxyl, salts with pharmaceutically acceptable bases, both inorganic and organic, such as, for example, alkali or alkaline earth hydroxides, ammonium hydroxide, amine, also heterocyclic.

The first group of particularly preferred compounds includes:

7-methoxyiminomethylcamptothecin (CPT 179);

7-methoxyiminomethyl-10-hydroxycamptothecin (CPT 211);

7-t-butoxyiminomethylcamptothecin (CPT 184);

7-t-butoxyiminomethyl-10-hydroxycamptothecin (CPT 212);

7-t-butoxyiminomethyl-10-methoxycamptothecin (CPT 220);

7-triphenylmethoxyiminomethylcamptothecin (CPT 192);

7- (2-amino) ethoxyiminomethylcamptothecin (CPT 188);

7-benzyloxyiminomethylcamptothecin (CPT 172);

7-phenoxyiminomethylcamptothecin (CPT 223);

7-p-methylbenzyloxyiminomethylcamptothecin (CPT 178);

7-pentafluorobenzyloxyiminomethylcamptothecin (CPT 182);

7-p-phenylbenzyloxyiminomethyl camptothecin (CPT 187);

7-oxiranylmethoxyiminomethylcamptothecin (CPT 213);

7- [2- (1-uracil) ethoxy] iminomethylcamptothecin (CPT 199);

7- (4-pyridyl) methoxyiminomethylcamptothecin (CPT 189);

7 - [(N-methyl) -4-piperidinyl] methoxyiminomethyl camptothecin (CPT 190);

7- (2- (4-morpholininyl] ethoxy] iminomethylcamptothecin (CPT 210);

PL 222 208 B1

A second group of particularly preferred compounds includes: 7- (6-D-glucosyloxy) iminomethylcamptothecin (CPT 216);

In a first preferred embodiment, there is provided a camptothecin N-oxide of general formula (I) wherein n is 1; and R4 is alkyl or aryl as defined above.

Among them, the more preferred compounds are:

7- (t-butoxy) iminomethylcamptothecin (CPT 184) of the formula

and 7-benzyloxyiminomethylcamptothecin (CPT 172)

The compounds of formula (I) can be prepared in various ways according to the nature of the R4 group and the presence of the oxygen linked to the nitrogen of the 7 iminomethyl group.

Compounds of formula (I) wherein n is 1 and R4 is as defined above, except aroyl and arylsulfonyl, can be prepared starting from 7-aldehyde-camptothecin (formula Ia, R5 hydrogen) or 7-keto-camptothecin (formula Ia) , R5 other than hydrogen).

Wherein R1 is the group -C (R5) = O, wherein R5 is as defined in formula (I), R2 and R3 are as defined in formula (I). Compounds of formula (Ia) are reacted with a compound of formula (IIa) R4O-NH2 wherein R4 is as defined above to give compounds of formula (I) wherein R1 is -C (R5) = N-OR4 , R4 is as defined in formula (I), except for aroyl and arylsulfonyl. Ejection can be carried out by well-known conventional methods, including normal oxime formation. Preferably, the molar ratio between 7-aldehyde or 7-keto camptothecin and hydroxylamine is comprised between 1: 3 and 3: 1. The hydroxylamine salts in question may be used. The reaction is carried out in the presence of a base, for example an inorganic base such as potassium carbonate, or an organic base such as triethylamine or diazabicyclononene, using polar solvents, preferably methanol or ethanol, and carrying out the reaction at a temperature between room temperature and the boiling point of the solvent used, optionally in the presence of dehydrating agents, for example sodium or magnesium sulfate, molecular sieves. If desired, it is also possible to carry out the reaction in the presence of a catalyst, for example a Lewis acid.

Alternatively, the above compounds can be prepared from camptothecin 7-aldehyde oxime (prepared as described by Sawada et al., Farm Chem. Bull. 39, (10) 2574 (1991)), or 7-keto by reaction with halide R4-X wherein X is preferably iodine, in a polar solvent, for example tetrahydrofuran or alcohols, and in the presence of a base, for example sodium hydride or carbonate.

Compounds of formula (I) in which n is 1 and R4 is aroyl or arylsulfonyl as defined for formula (I) can be prepared starting from camptothecin 7-oxime, the preparation of which is described in the previous paragraph, with acyl chloride R4-COCl, in polar solvents, and in the presence of a base, preferably pyridine, or directly in pyridine as described by Cho et al., J. Org. Chem. 62, 2230 (1997).

Compounds of formula (I) in which n is 0 and R4 is as defined above, except aroyl, can be obtained starting from 7-aldehyde-camptothecin (formula Ia, R5 = hydrogen) or 7-ketocamptothecin (formula Ia, R5 other than hydrogen).

wherein R1 is the group -C (R5) = O, where R5 is as defined in formula (I), R2 and R3 are as defined in formula (I). The compound of formula (Ia) reacts with a compound of formula (IIb) R ₄ -NH ₂ , wherein R ₄ is as defined above, to obtain compounds of formula (I), wherein R ₁ is a group -C (R ₅ ) = ₄ , R ₄ is as defined in formula 1, except for aroyl.

The reaction can be carried out by well-known conventional methods, including normal imine formation. Preferably, the molar ratio between 7-aldehyde or 7-keto camptothecin and the amine is between 1: 3 and 3: 1: The salts of the amine in question can be used. The reaction is carried out in the presence of a base, for example an inorganic base such as potassium carbonate, or an organic base such as triethylamine or diazabicyclononene, using polar solvents, preferably methanol or ethanol, and carrying out the reaction at a temperature comprised between room temperature and the boiling point of the solvent, optionally in the presence of agents dehydrators, for example sodium or magnesium sulfate, molecular sieves. If desired, it is also possible to carry out the reaction in the presence of a catalyst, for example a Lewis acid (as described, for example, by Moretti and Torre, Synthesis, 1970, 141; or by Kobayashi et al., Synlett, 1977, 115).

7-aldehyde-camptothecin and 7-camptothecin oxime are described in patent application EP 0056692 and in the mentioned publication by Sawada et al., Chem. Farm. Bull. 39, (10) 2574 (1991).

PL 222 208 B1

The N1-oxides of compounds of formula (I) were prepared according to well-known methods for the oxidation of heteroaromatic nitrogen, preferably by oxidation with acetic or trifluoroacetic acid and hydrogen peroxide, or by reaction with organic peroxyacids (A. Albini and S. Pietra, Heterocyclic N-oxides, CRC , 1991).

Regarding the meanings of R4 present in the various reagents of formula II, these reagents are commercially available or can be prepared according to methods well known in the literature, supplemented by the knowledge of those skilled in the art.

Pharmaceutically acceptable salts are prepared according to methods well known in the literature which need not be disclosed in further detail.

The compounds described in the present invention exhibit antiproliferative properties, therefore are useful for their therapeutic activity, and possess physico-chemical properties that enable them to be formulated into pharmaceutical compositions.

The pharmaceutical compositions contain at least the compound of formula (I) in an amount sufficient to achieve a significant therapeutic effect, especially an anti-tumor effect. The compositions encompassed by the present invention are conventional and are prepared by conventional methods used in the pharmaceutical industry. According to the desired route of administration, the compositions should be in solid or liquid form suitable for oral, parenteral, intravenous administration. The compositions of the present invention together with the active ingredients contain at least a pharmaceutically acceptable vehicle or excipient. Co-adjuvants of the compositions, for example, solubilizing, dispersing, suspending, and emulsifying agents, may be especially useful.

The compounds of formula (I) may be used together with other active ingredients, for example other anti-cancer drugs, both in separate and single dosage forms.

The compounds of the present invention are useful drugs with antitumor activity, for example in lung tumors such as non-small cell lung cancer, colorectal tumors, prostate, gliomas.

The cytotoxic activity of the compounds of the present invention was tested in the cellular systems of human cancer cells using anti-proliferative assays as a method to evaluate the cytotoxic potential.

The cell line used is non-small cell lung cancer, which belongs to the non-small cell histotype, called NCI H460.

The preferred compounds of 7- (t-butoxyiminomethyl camptothecin (CPT 184) and 7-benzyloxyiminomethyl camptothecin (CPT 172) were tested by comparing them with Topotecan (TPT), and with 7-hydroxyiminomethyl camptothecin (CPT 181) disclosed by Sawada et al. In Farm Chem. 39 (10), 2574-2580, (1991), which is the closest structural analog to the compounds of formula (I) of the present invention.

For in vivo studies, solubilization was performed in 10% DMSO in double-distilled, non-saline water, and administration by the oral route was carried out in a volume of 10 ml / kg.

Antitumor effect

Atimic nu / nu Swiss mice (Charles River, Calco, Italia), 10-12 weeks old, were used for the study. The animals were housed in laminar flow rooms according to the guidelines of the United Kingdom Co-ordination Committee Cancer Research. The experimental protocols were provided by the Ethics Committee for Animal Research Istituto Nazionale per Io Studio e Cura dei Tumori.

Tumor fragments with dimensions 2x2x2 mm, obtained from mice inoculated sc 10 ⁶ cells NCI H460 / topo, were implanted sc bilaterally in groups of 5 mice each.

The animals were treated with the compounds when the tumor was palpable according to the following schedule:

CPT172 (8 mg / kg, po) q4dx4

CPT172 (16 mg / kg, po) q4dx4

CPT172 (24 mg / kg, po) q4dx4

CPT172 (2 mg / kg, po) qdx5x10w

CPT181 (15 mg / kg, po) q4dx4

CPT181 (25 mg / kg, po) q4dx4

CPT184 (2 mg / kg, po) q4dx4

CPT 184 (5 mg / kg, po) q10dx6

Topotecan (15 mg / kg, po) q4dx4

Topotecan (10 mg / kg, po) q4dx4

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Twice a week, the width, minimum diameter (1), length and maximum diameter (L) of the tumors, in mm, were measured using a Vernier tumor measuring instrument. Tumor volume (mm ³ ) was calculated according to the formula ¹² × L / 2. The efficacy of the compounds was assessed as% TVI of the treated group versus the control group according to the formula TVI% = 100- (T / Cx1 00), where T is the mean value of the tumor volume of the treated group and C is the control group. The compound is considered active when TVI% = 50.

Table 1 below gives the experimental results.

T A B E L A 1

Antitumor activity of camptothecin analogues in the treatment of NCI H460 lung cancer

Relationship Dose (mg / kg p.o.) Scheme treatment Effectiveness (TVI%) Toxicity Mortality Weight Loss (%) CPU172 8 q4dx4 77 16 q4dx4 88 24 q4dx4 97 0/4 6 2 qdx5x10w 90 0/3 0 CPT181 15 q4dx4 40 0/4 0 25 q4dx4 70 0/4 0 CPU184 2 q4dx4 100 0/5 0 5 q10dx6 99 0/4 9 TPT q4dx4 94 0/4 0 q4dx4 89 0/5 10 q4dx4 64 0/5 0

TVI% was assessed 5-10 days after the last treatment

CPT172 is effective at a variety of doses and treatment regimens; CPT 184 appears to be a very active compound in low doses and with various treatment regimens, respectively, both compounds are particularly promising compounds for clinical applications.

Further advantages of these compounds can be identified over a wide range of effective doses, indicating an increase in the therapeutic index and easier handling in therapeutic application; especially if prolonged administration is envisaged, primarily in injectable preparations, using different dosages and schedules. In such applications, CPT 172 proves to be more advantageous in terms of reduced toxicity.

A significant disadvantage of conventional camptothecins is the reversibility of their binding in the tertiary complex (drug-DNA-enzyme). This reversibility affects the efficacy as it prevents transformation of single DNA strand cleavage into DNA double strand cleavage during DNA synthesis.

Table 2 below indicates the maintenance of DNA cleavage to a selected number of in-vitro cleavage sites. After the drug had been incubated for 20 minutes in the reaction mixture containing labeled DNA and purified enzyme, sodium chloride (0.6 M) was added to aid dissociation of the tertiary complex.

The result shown in the table as the percentage of DNA cleft retention at sites, tested after approximately 10 min., Is indicative of near complete reversibility of clefts for camptothecin and Topotecan, and clear maintenance for CPT 172 and CPT 184.

PL 222 208 B1

T a b e l a 2

Maintenance of DNA cleavage stimulated by camptothecin caused by topoisomerase and at selected sites

LEK (10 im) Holding (%) Camptothecin 16 Topotecan 16 CPT 181 28 CPT 184 72 CPT 172 80

The advantage of the compounds of the present invention is apparent in overcoming the limitation of the reversibility of the tertiary complex over the prior art.

In preclinical studies, CPT 184 exhibits cytotoxic activity against a variety of tumor cell lines.

This broad spectrum of antitumor activity has been confirmed in mice transplanted with human cancer xenografts, including NSCLC (H460, A549), prostate cancer (JCA-1), glioblastoma (GBM / 7), gastric cancer (MKN28), osteosarcoma (U20S), ovarian cancer (A2780 / Dx, A2780 / DDP) and colon (HT29, CoBA) as well as murine lung cancer (M109) and a leukemia model (L1210).

Preclinical data suggest that CPT 184 may be an active antitumor agent against human tumors, particularly against non-small cell lung cancer (NSCLC), glioblastoma, and prostate cancer (Table 3).

T a b e l a 3

Antitumor activity of CPT 172, CPT 184 vs TPT in various tumor models

CPT184 CPT172 TPT 2 mg / kg 3 mg / kg 20 mg / kg 25 mg / kg 15 mg / kg H460 99 97 98 (5/8 *) HT29 92 88 65 CoBA 84 87 84 93 85 GBM / 7 97 98 93 98 96 (3/10 *) (2/8 *) (3/11 *) (9/12 *) (1/10 *) U87 80 87 82 A2780 100 - 96 (1/8 *) A2789 / Dx 92 100 92 (1/8 *) (8/8 *) (2/10 *) A2780 / DDP 77 90 - 92 IGROV-1 93 96 - 89 JCA-1 98 98 - 95 (5/8 *) DU145 95 77 L1210 > 400 * * * > 400 ** 39

PL 222 208 B1

Results are expressed for solid tumors as% TVI = tumor volume inhibition = 100- (treated group tumor weight / - average control tumor weight x 100) and for L1210 as% ILS = percent life span increase [(treated group MST / control MST ) x 100] - 100.

* no tumor was noticed at the end of treatment for approximately 10 days from the last administration.

**> 50% and ***> 80% cured live 120 d mice after leukemia injection.

H460 = NSCLC

HT29 and CoBA = colon cancer

IGROV-1, A2780, A2780 / Dx, and A2780 / DDP = ovarian cancer

GBM / 7 and U87 = glioblastoma

JCA-1 and DU145 - prostate cancer

L1210 = murine leukemia

The high cytotoxic potency of the compounds of the invention, represented here experimentally with one of the preferred compounds, CPT 184, is also reflected by the potent anti-tumor activity. Using a set of tumor xenografts with remarkable reactivity to Topotecan (TPT) (i.e., TVI> 89%), the spectrum of antitumor activity of CPT 184 was broadly improved with the compounds of the invention against a large number of human tumor models.

In particular, impressive anti-tumor efficacy has been found in the treatment of multiple tumor models where complete regressions have been achieved in a large number of treated animals. Furthermore, the compounds of the invention, especially CPT 184, were able to induce 100% CR in A2780 and DX tumors characterized by the MDR phenotype. This observation is very important, indicating that the compounds of the present invention are not a substrate for P-glycoproteins. Additional therapeutic advantages of the compounds of the present invention relate to:

a) improvement of the therapeutic index,

b) the effectiveness of the drug in a wide range of doses,

c) evidence of efficacy using completely different regimens, making the compounds of the present invention less regimen dependent than Topotecan.

The following Examples illustrate the invention.

P r z k ł a d 1

7-benzyloxyiminomethylcamptothecin (CPT 172)

500 mg (1.33 mmol) of 7-formylcamptothecin was dissolved in 100 ml of ethanol, 15 ml of pyridine and 638 mg (4 mmol) of O-benzylhydroxylamine hydrochloride were added and the mixture was kept at reflux for 5 hours. The solvent was evaporated in vacuo, and the residue thus obtained was purified by flash chromatography on silica gel using hexane / ethyl acetate 4/6 as the eluent.

Yield 65%, mp: 200 ° -205 ° C dec.

The resulting product is an approximately 8: 2 mixture of syn and anti isomers (isomer A: Rf 0.32; isomer B, Rf: 0.19 on Merck 60 F254 silica gel, eluent hexane / ethyl acetate 3/7).

HPLC: Analyzes were performed on an instrument equipped with a quad pump (HP 1050) with a Rheodyne injector (20 µΐ loop) and a diode array detector (Hp 1050) controlled by the HPLC-ChemStation software. Detection of the spectrum was done from 200 to 600 nm and chromatograms were recorded at 360 and 400 nm.

A reversed-phase C18 column (Rainin C18; 25 x 0.4 cm, Varian) with an RP precolumn was used. The analysis was performed with a linear elution gradient starting from a mixture of acetonitrile: water 30:70 to acetonitrile 100% in 20 min with a flow of 1 ml / min. The retention times were: 12.51 min for the B isomer and 14.48 min for the A isomer.

¹ H-NMR (300 MHz; DMSO-d6): δ 0.88 (t, H3-18A + H3-18B), 1.87 (m, H2-19A + H2-19B), 5.18 (s, H2-5B), 5.21 (s, H2-FB), 5.30 (H2-FA), 5.40 (s, H2-5A), 5.45 (s, H2-17A + H2-17B) , 6.53 (s; OHA + OHB), 7.3-7.6 (m, ArA + ArB + H-14A + H-14B), 7.75 (m, H-11A + H-11B), 7 , 85-7.95 (m, H10A + H-10B), 7.98 (dd, H-12B), 8.18-8.27 (m, H-12A + H9-B), 8.45 ( s, CH = NB), 8.59 (dd, H-9A), 9.38 (s, CH = NA).

Mass analysis m / z 481 (M + 100) 374 (30) 330 (70) 300 (30) 273 (20) 243 (20) 91 (34).

P r z k ł a d 2

7-t-butoxyiminomethylcamptothecin (CPT 184)

400 mg (1.06 mmol) of 7-formylcamptothecin was dissolved in 80 ml of ethanol, 12 ml of pyridine, 400 mg (3.18 mmol) of O-t-butylhydroxylamine hydrochloride were added and the mixture was allowed to stand in the water.

Hours at reflux. The solvent was evaporated in vacuo and the residue was purified by flash chromatography on silica gel using hexane / ethyl acetate 4/6 as eluent. 322 mg (0.72 mmol) of a yellow solid are obtained. Yield 68% mp: 250 ° C dec.

The resulting product was an approximately 8: 2 mixture of the two syn and anti isomers (isomer A: Rf 0.31; isomer B. Rf: 0.24 on Merck 60 F254 silica gel, eluent hexane / ethyl acetate 3/7).

HPLC: Analyzes were performed on an instrument equipped with a quad pump (HP 1050) with a Rheodyne injector (20 µΐ loop) and a diode array detector (Hp 1050) controlled by the HPLC-ChemStation software. Detection of the spectrum was done from 200 to 600 nm and chromatograms were recorded at 360 and 400 nm.

A reversed-phase C18 column (Rainin C18; 25 x 0.4 cm, Varian) with an RP precolumn was used. The analysis was performed with a linear gradient of elution starting from a mixture of acetonitrile: water 30:70 to acetonitrile 100% in 20 min with a flow of 1 ml / min. The retention times were: 12.92 min for isomer B and 14.61 for isomer A.

¹ H-NMR (300 MHz; DMSO-da): δ: 0.88 (t, H3-18A + H3-18B), 1.30 (s, tbut. B), 1.47 (s, t -but A) 1.87 (m, H2-19A + H2-19B) 5.18 (s, H2-5B), 5.37 (H2-5A), 5.42 (s, H2-17A + H2 -17B), 6.54 (s, OHA + OHB), 7.35 (s, H-14A), 7.36 (s, H-14B) 7.69-7.83 (m, H-11A + H-11B), 7.85-7.98 (m, H-10A + H-10B), 8.07 (dd, H-9B), 8.16-8.27 (m, H-9A + H -12B) 8.40 (s, CHB), 8.62 (dd, H-12A), 9.31 (s, CHA).

Mass analysis m / z 448 (M + 28) 391 (40) 374 (100) 362 (40) 330 (34) 57 (17).

The following compounds were prepared according to the same procedure:

7-t-butoxyiminomethyl-10-hydroxycamptothecin (CPT 212); mp 195 dec.

¹ H-NMR (DMSO-d6) δ = 0.88 (t, J = 7.35 Hz, H3-18) 1.45 (s, 3 -CH3) 1.80-1.90 (m, H2- 19) 5.12 (s, H2-5 anti) 5.33 (s, H2-5 syn) 5.45 (m, H2-17 syn; H2-17 anti) 6.50 (s, -OH) 7 . 25 (d, J = 2.57 Hz, H-9 anti) s 7.30 (s, H-14 syn; H-14 anti) 7.43-7.50 (m, H-11 syn; H -11 anti) 7.70 (d, J = 2.57 Hz, H-9 syn) 8.15 (d, J = 9.19 Hz; H-12 syn, H-12 anti) 8.25 (s , -CH = N anti) 9.00 (s, -CH = N syn).

Mass analysis m / z: 463 (M + 16) 419 (15) 407 (25) 390 (43) 346 (100) 318 (10).

7-t-butoxyiminomethyl-10-methoxycamptothecin (CPT 220); mp: 250 ° C dec.

¹ H-NMR (DMSO-d6) δ: 0.88 (t, J = 7.35 Hz, H3-18) 1.47 (s, 3 - CH3) 1.80-1.93 (m, H2- 19) 3.95 (s, -OCH3 anti) 3.98 (s, -OCH3 syn) 5.17 (s, H2-5 anti) 5.30-5.45 (m, H2-5 syn; H2- 17 syn; H2-17 anti) 6.50 (s, -OH) 7.29 (s, H-14) 7.56 (dd, J = 9.19 Hz; J = 2.57 Hz; H-11 ) 7.90 (d, J = 2.57 Hz; H-9) 8.12 (d, J = 9.19 Hz; H-12) 8.39 (s, -CH = N anti) 9.33 (s, -CH = N syn).

Mass analysis m / z: 477 (M56, M) 421 (74) 404 (100) 392 (66) 360 (18.5) 303 (6) 274 (7.5).

7-p-nitrobenzyloxyiminomethylcamptothecin (CPT 177);

7-p-methylbenzyloxyiminomethylcamptothecin (CPT 178) mp. 203 ° C dec.

7-methoxyiminomethylcamptothecin (CPT 179) mp. 230 ° C dec.

7-methoxyiminomethyl-10-hydroxycamptothecin (CPT 211); s mp: 268 ° C dec.

¹ H NMR (DMSO-d6) δ = 0.87 (t, J = 7.35 Hz, H3-18) 1.80-1.90 (m, H2-19) 4.13 (s, -OCH3) 5.32 (s, H2-5) 5.41 (s, H2-17) 6.50 (s, -OH) 7.26 (s, H-14) 7.47 (dd, J = 9.19 Hz; J = 2.56 Hz, H-11) 7.75 (d, J = 2.56 Hz, H-9) 8.08 (d, J = 9.19 Hz, H-12) 9.04 (s, -CH = N).

7-pentafluorobenzyloxyiminomethylcamptothecin (CPT 182) mp. 200 ° C dec.

7-carboxymethoxyiminomethylcamptothecin (CPT 183);

7- (carboxydimethylmethoxy) iminomethylcamptothecin; mp: 193 ° C dec.

¹ H NMR (CDCl3) δ = 1.02 (t, J = 7.35 Hz, H3-18) 1.69 (s, -CH3), 1.72 (s, -CH3), 1,81-1,95 (m, H2-19) 3.60 (s, -OH) 5.24 (d, J = 16.55 Hz, H-17A) 5.32 (s, Hz-5) 5.65 (d, J = 16.55 Hz, H-17B) 7.64 (s, H-14) 7.67 (ddd, J = 6.99 Hz; J = 8.47 Hz; J = 1.47 Hz, H-11 ) 7.80 (ddd, J = 6.99Hz; J = 8.47Hz; J = 1.47Hz, H-10) 8.10-8.16 (m, H-9; H-12) 9.10 (s, -CH = N).

7- (tert-butoxycarbonyl-2-propoxy) iminomethylcamptothecin (CPT 224); mp: 180 ° C dec.

¹ H NMR (DMSO-d6) δ = 0.88 (t, J = 7 Hz, H3-18) 1.44 (s, 3 -CH3), 1.60 (s, 2 -CH3) 1,80- 1.92 (m, H2-19) 5.27 (s, H2-5) 5.43 (s, H2-17) 6.53 (s, -OH) 7.35 (s, H-14) 7 .76 (ddd, J = 8.46 Hz; J = 8.46 Hz; J = 1.47 Hz, H-11) 7.92 (ddd, J = 8.46 Hz; J = 8.46 Hz; J = 1.47 Hz, H-10) 8.23 (dd, J = 8.46 Hz; J = 1.47 Hz, H-12) 8.65 (dd, J = 8.46 Hz; J = 1.47 Hz, H-9) 9.20 (s, - CH = N). Mass analysis m / z: 534 (M + 13) 477 (29) 374 (55) 273 (10) 57 (100) 41 (57).

7-p-phenylbenzyloxyiminomethyl camptothecin (CPT 187) mp. 200-202 ° C dec.

7-oxiranylmethoxyiminomethylcamptothecin (CPT 213);

¹ H NMR (CDCl3) δ = 0.87 (t, J = 7 Hz, H3-18), 0,80-2,00 (m, J = 7 Hz, H2 - 19) 2.80 (1H, m, -CH2O) 3.05 (1H, m, -CH2O) 3.40 (m, -CH-O) 3.75 (s, -OH) 4.30 (1H, m, -CH2-ON) 4.73 (1H, m, -CH2-ON)

PL 222 208 B1

5.33 (d, J = 16Hz, H-17A) 5.45 (s, H2-5) 5.75 (d, J = 16Hz, H-17B) 7.70 (s, H-14) 7.75 (m, H-11) 7.85 (m, H-10) 8.15-8.35 (m, H-9; H-12) 9.12 (s, -CH = N)

7- (2-amino) ethoxyiminomethylcamptothecin (CPT 188); mp 220 ° C dec.

7- (4-pyridyl) methoxyiminomethylcamptothecin (CPT 189) mp. 220 ° C dec. mass analysis m / z M + 482

7 - [(N-methyl) -4-piperidinyl] methoxyiminomethyl camptothecin (CPT 190) mp. 185-190 ° C dec. mass analysis m / z M + 502

7-ethoxyiminomethylcamptothecin;

7-isopropyloxyiminomethylcamptothecin

7- (2-methylbutoxy) iminomethylcamptothecin;

7-cyclohexyloxyiminomethylcamptothecin;

7-cyclohexylmethoxyiminomethylcamptothecin;

7-cyclooctyloxyiminomethylcamptothecin;

7-cyclooctylmethoxyiminomethylcamptothecin;

7- (1-adamantyloxy) iminomethylcamptothecin;

7- (1-adamantylmethoxy) iminomethylcamptothecin;

Fenoksyiminometylokamptotecyna 7- (CPT 223) ¹ H NMR (DMSO-d6) δ = 0.89 (t, J = 7.35 Hz, H3-I8), 1,81-1,95 (m, H-19), 5 25 (s, H2-5 anti) 5.42 (s, H2-17anti) 5.45 (s, H2-5 syn) 5.52 (s, H2-17 syn) 6.56 (s, -OH) 7.1 S, 7.55 (m, 5Ar; H-14) 7.83 (m, H-11) 7.96 (m, H-10) 8.28 (dd, J = 8.09 Hz; J = 1.10 Hz, H-12) 8.73 (dd, J = 8.09 Hz; J = 1.10 Hz, H-9) 8.92 (s, -CH = N anti) 9.84 (s, -CH = N syn).

mass analysis m / z: 467 (M + 33) 373 (100) 329 (62) 314 (72) 273 (62) 244 (52) 135 (38) 57 (25) 43 (39).

7- (2-naphthyloxy) iminomethylcamptothecin;

7- (9-anthrylmethoxy) iminomethylcamptothecin;

7- [2- (2,4-difluorophenyl) ethoxy] iminomethylcamptothecin;

7- (4-t-butylbenzyloxy) iminomethylcamptothecin;

7-triphenylmethoxyiminomethylcamptothecin (CPT 192) mp. 140 ° C dec .;

7- (2-N, N-dimethylaminoethoxy) iminomethylcamptothecin (CPT 197);

7- [N- (4-aminobutyl) -2-aminoethoxy] iminomethylcamptothecin;

7- [N- [N- (3-amino-1-propyl) -4-amino-1-butyl] -3-aminopropoxy] iminomethylcamptothecin;

7- [2- (1-uracil) ethoxy] iminomethylcamptothecin (CPT 199); mp: 197-200 ° C dec.

¹ H NMR (DMSO-d6) δ = 0.88 (t, J = 7.35 Hz, H3-18) 1.80-1.95 (m, H2-19) 3.90 (t, J = 6 Hz, -CH2N anti) 4.15 (t, J = 6 Hz, -CH2N syn) 4.35 (t, J = 6 Hz, -CH2O anti) 4.58 (t, J = 6 Hz, -CH2O syn ) 5.00 (d., J = 8 Hz, H-5 U anti) 5.35-5.50 (m, H2-5 anti; H2-5 syn; H2-17 anti; H2-17 syn) 5 , 55 (d, J = 8 Hz, H-5 U syn) 6.55 (s, -OH) 7.15 (d, J = 8 Hz, H-6 U anti) 7.40 (s, H- 14) 7.64 (d, J = 8 Hz ;, H-6 U syn) 7.70-7.82 (m, H-10 syn; H-10 anti) 7.85-8.00 (m, H-11 syn; H-11 anti; H-12 anti) 8.23 (m, H-12 syn; H-9 anti) 8.48 (s, -CH = N anti) 8.60 (dd, J = 8.46 Hz; J = 1.47 Hz, H-9 syn) 9.35 (s, -CH = N syn) 11.3 (br s, NH U).

7- [2- (4-morpholininyl] ethoxy] iminomethylcamptothecin (CPT 210), mp: 158-160 ° C dec.

¹ H NMR (CDCl3) δ = 1.06 (t, J = 7.35 Hz, H3-18) 1.84-2.00 (m, H2-19) 2.62 (t, J = 4.78 Hz, -CH2-N morph) 2.87 (t, J = 5.52 Hz, -CH2-N) 3.60 (s, -OH) 3.79 (t, J = 4.78 Hz, - CH2O morph.) 4.59 (t, J = 5.52 Hz, -CH2-O) 5.33 (d, J = 16.18 Hz, H-17A) 5.45 (s, H2-5) 5 .77 (d, J = 16.18 Hz; H-17B) 7.69 (s, H-14) 7.73 (ddd, J = 1.47 Hz; J = 8.46 Hz; J = 8, 46 Hz; H-11) 7.87 (ddd, J = 1.47 Hz; J = 8.46 Hz; J = 8.46 Hz, H-10) 8.19-8.31 (m, H- 9; H-12) 9.12 (s, -CH = N).

Mass analysis m / z: 504 (M + 4) 373 (23) 329 (26) 272 (18) 244 (20) 216 (13) 100 (100).

7- (6-uracil) methoxyiminomethylcamptothecin;

7- (4-hydroxybutoxy) iminomethylcamptothecin;

7- (2-thienyl) methoxyiminomethylcamptothecin;

7- (4-thiazolyl) methoxyiminomethylcamptothecin;

7- (6-D-galactosyloxy) iminomethylcamptothecin;

7- (6-D-glucosyloxy) iminomethylcamptothecin;

7- (6-D-glucosyloxy) iminomethylcamptothecin (CPT 216); mp: 210 ° C dec.

¹ H NMR (DMSO-d6) δ = 0.85 (t, J = 7.3 Hz, H3-18) 1.75-1.95 (m, H2-19), 3,50-5,00 (m , 10H galactic) 5.35 (s, H2-5) 5.45 (s, H2-17) 6.25 (d, -OH galactic) 6.55 (s, -OH) 6.65 (d , -OH gaIact) 7.35 (s, H14) 7.80 (m, H-10) 7.98 (m, H-11) 8.25 (dd, J = 8.47 Hz; J = 1.46 Hz, H-12) 8.60 (dd, J = 8.47 Hz; J = 1.46 Hz, H-9) 9.35 (s, -CH = N).

PL 222 208 B1

7- (1,2: 3,4-di-O-isopropylidene-D-galactopyranosyloxy) -iminomethylcamptothecin (CPT 215);

¹ H NMR (DMSO-d6) δ = 0.87 (t, J = 7.30 Hz, H3-18) 1.30-1.45 (m, 4 -CH3), 3,90-4,70 (m , H2-6 ';H-5'; H-4 ';H-3'; H-2) 1.80-1.93 (m, H2-19) 5.35 (s, H2-5) 5 , 45 (s, H2-17) 5.60 (d, J = 5.52Hz, H-1) 6.52 (s, -OH) 7.35 (s, H-14) 7.75 (m , H-10 syn; H-10 anti) 7.90 (m, - H-11 syn; H-11 anti) 8.05 (dd, J = 8.47 Hz; J = 1.47 Hz, H- 12 anti) 8.20 (m, H-12 syn; H-9 anti) 8.50 (s, -CH = N anti) 8.65 (dd, J = 8.47 Hz; J = 1.47 Hz ; H-9 syn) 9.40 (s, -CH = N syn).

Mass analysis m / z: 634 (M + 1 13) 576 (10) 486 (18) 347 (35) 329 (45) 314 (49) 302 (28) 246 (100) 242 (55) 187 (26).

7- (1-benzyloxyimino) ethylcamptothecin (CPT 186);

7- [1 (-t-butoxyimino) ethyl] camptothecin.

P r z k ł a d 3

7-benzoylcamptothecin (CPT 170)

Concentrated sulfuric acid (0.17 ml) and benzaldehyde (304 mg, 2.87 mmol) were added dropwise to a suspension of camptothecin (200 mg, 0.57 mmol) in CH3COOH (0.8 ml) and water (0.8 ml). The reaction mixture was cooled to 0 ° C and 80% t-butyl peroxide (128 mg, 1.14 mmol) and a solution of FeSO4 (317 mg, 1.14 mmol) in water (0.56 mL) were added sequentially. After stirring overnight at room temperature, water was added, and the resulting precipitate was filtered off with suction. The mother liquors were reacted with methylene chloride (3 times); the organic phases were dried over Na2SO4, filtered and evaporated in vacuo. The solid product was collected with the precipitate previously separated. The product was purified by flash chromatography on silica gel, eluting with a 98/2 methylene chloride / methanol mixture. 90 mg (0.2 mmol) of the product are obtained. Yield 35%.

¹ H-NMR (300 MHz; DMSO-d6): δ: 0.9 (t, 3H H3-18), 1.85 (m, 2H, H2-19), 5 (s, 2H, H2-5) , 5.4 (2H, H2-5), 5.4 (s, 2H H2-17), 6.6 (s, -1H OH), 7.4 (s, 1H, H14), 7.55- 7.85 (m, 5H, H1-10, H11.3Ar), 7.95-8 (m, 3H-H12 2Ar), 8.3 (dd, 1H-H-9).

P r z k ł a d 4

7- [α- (hydroxyimino) benzyl] camptothecin (CPT 185);

A solution of 7-benzoyl camptothecin (50 mg, 0.11 mmol), hydroxylamine hydrochloride (24 mg, 0.33 mmol), pyridine (1.4 ml) in 10 ml of ethanol was prepared and allowed to reflux for 24 hours. The solvent was stripped off in vacuo. The product was purified by flash chromatography on silica gel using methylene chloride / methanol 98/2 as eluent. 25 mg of a yellow solid was obtained. Yield 48%.

The obtained product was a mixture of the two syn and anti isomers (isomer A: Rf 0.35; isomer B, Rf: 0.31 on Merck 60 F2s4 silica gel, eluent methylene chloride / methanol 95/5).

¹ H-NMR (300 MHz; DMSO-d6): δ: 0.9 (t, H3-18A + H3-18B), 1.86 (m, H2-19A + H2-19B) 4.8 (m, H2-5 A + H2-5B), 5.85 (s, H2-17A + H2-17B); 6.55 (s, -OH B), 7.60 (s OH A), 7.35-7.55 (m, Ar A + Ar B + H10A + H-1 OB + H-11A + H-11 B + H-14A + H-14B) 7.6-7.7 (m, H-12A + H-12B)

P r z k ł a d 5

7-Phenylamino-methylcamptothecin (CPT 154)

100 mg (0.26 mmol) of 7-formylcamptothecin dissolved in 20 ml of methylene chloride and 2.5 ml (0.26 mmol) of aniline dissolved in 0.5 ml of methylene chloride were added to the ytterbium triflate suspension (16.5 mg, 0.026 mmol). , 10% mol) in 5 ml of methylene chloride containing MS 4A and left for 3.5 hours while stirring at room temperature, then the solvent was evaporated in vacuo. The product was purified by flash chromatography on silica gel using methylene chloride / methanol 98/2 as eluent. 60 mg of a yellow solid was obtained.

51% yield. mp: 255-258 ° C dec.

¹ H-NMR (300 MHz; DMSO-d6): δ: 0.8 (t, 3H H3-18), 1.75 (m, 2H, H2-19), 5.35 (s, 2H, H2- 5), 5.5 (s, 2H H2-17), 6.45 (s, -1H OH), 7.25-7.35 (m, 2H H1-Ar + H-14), 7.4- 7.5 (m, 4H, Ar), 7.75 (1H, ddd, H-11) 7.85 (ddd, 1H-H10), 8.2 (dd, 1H-H-12) 8.9 (dd , 1H, H-9), 9.6 (s, 1H, CH = N).

The following compounds were prepared using the same procedure:

7-cyclohexyliminomethylcamptothecin (CPT 156);

7-p-nitrophenyliminomethylcamptothecin (CPT 160), mp. 260-265 ° C dec .;

7- (4-hydroxy) butyliminomethylcamptothecin (CP 169) mp. 140 ° C dec .;

7-dimethylaminoethyliminomethylcamptothecin (CPT 171);

7-benzyliminomethylcamptothecin (CPT 175);

7-t-butyliminomethylcamptothecin;

7-allyliminomethylcamptothecin;

PL 222 208 B1

7- (2-thienyl) iminomethylcamptothecin;

7- (4-amino) butyliminomethylcamptothecin;

7- (3-aminopropyl-4-aminobutyl-3-aminopropyl) iminomethylcamptothecin;

7- (2-anthrylmethyl) iminomethylcamptothecin;

7- (6-D-galactosyl) iminomethylcamptothecin;

7- (2-quinolylmethyl) iminomethylcamptothecin.

P r z k ł a d 6

7- (benzoylaryliminomethylmethyl) camptothecin (CPT 191)

A solution of benzoyl chloride (0.16 ml, 1.4 mmol) in 5 ml of pyridine was prepared, 500 mg (1.3 mmol) of 7-hydroxyiminomethyl camptothecin was added and allowed to stir overnight at room temperature. After evaporating the pyridine in vacuo, sodium bicarbonate solution was added and extraction was carried out three times with methylene chloride. After drying with sodium sulfate and filtration, the solvent was evaporated. The product was purified by flash chromatography on silica gel using methylene chloride / methanol 98/2 as eluent. 200 mg (0.04 mmol) of a yellow solid are obtained. 32% yield. mp: 210 ° C dec.

¹ H-NMR (300 MHz; DMSO-d6): δ: 0.8 (t, H3-), 1.8 (m, H2) 5.45 (s, H2-5), 5.55 (s, H2-17), 6.6 (s, 1H-OH), 7.3 (s, 1H, H-14), 7.75-8 (m, 5H H-10 + H-11 + 3Ar) 8.25 (m, 2H, 2Ar) 8.3 (dd, 1H, H-12) 8.75 (dd, 1H, H-9), 10.05 (s, 1H, CH = N).

The following compounds were prepared using the same procedure:

7-p-nitrobenzoyloxyiminomethylcamptothecin

7-p-cyanobenzoyloxyiminomethylcamptothecin

7-p-Tolylsulfonyloxyiminomethylcamptothecin

P r z k ł a d 7

7-t-butoxyiminomethyl camptothecin N-oxide (CPT 198)

7-t-butoxyiminomethylcamptothecin (30 mg, 0.067 mmol) was dissolved in acetic acid (5.2 ml) and 30% hydrogen peroxide was added. The mixture was heated at 70-80 ° C for 9 hours, concentrated to one third and the residue poured into ice water. The precipitated solid was collected by suction and purified by flash chromatography using a 1/1 mixture of hexane / ethyl acetate as the eluent. 7-t-butoxyiminomethyl camptothecin N-oxide was obtained as a yellow powder (15.5 mg). 50% yield. mp: 185-190 ° C dec

¹ H NMR (DMSO-d6) δ = 0.87 (t, J = 7 Hz, H3-18) 1.48 (s, 3 - CH3) 1,76-20 1.95 (m, H2-19) 5.37 (s, H2-5) 5.42 (s, H2-17) 6.60 (s, -OH) 7.85-8.00 (m, H-10; H-11) 8.15 (s, H-14) 8.65-8.75 (m, H-9; H12) 9.2 (s, -CH = N).

The following compounds were prepared according to the same procedure:

N-oxide 7-metoksyiminometylokamptotecyny (CPT 208) ¹ H NMR (DMSO-d6) δ = 0.87 (t, J = 7.35 Hz, H3-18), 1,78-1,93 (m, H2-19 ) 4.12 (s, -OCH3) 5.35 (s, H2-5) 5.43 (s, H2-17) 6.54 (s, -OH) 7.84-8.00 (m, H -10; H-11) 8.11 (s, H-14) 8.68-8.73 (m, H-9; H-12) 9.21 (s, -CH = N).

7- (carboxydimethylmethoxy) iminomethylcamptothecin N-oxide;

7- (hydroxymethyldimethylmethoxy) iminomethylcamptothecin N-oxide.

P r z k ł a d 8

7-p-nitrobenzyloxyiminomethylcamptothecin (CPT 177)

To a suspension of 7-hydroxyiminomethylcamptothecin (40 mg, 0.102 mmol) and sodium carbonate (10.9 mg, 0.102 mmol) in ethanol (4 ml), 4-nitrobenzyl bromide (22 mg, 0.102 mmol) was added and the mixture was refluxed for 2.5 hours The solvent was evaporated under reduced pressure. The residue was purified by chromatography using 3/7 hexane / ethyl acetate as eluent to give 10.5 mg of 7-p-nitrobenzyloxyiminomethyl camptothecin.

Yield: 20% ¹ H NMR (DMSO-d6) δ = 0.88 (t, J = 7 Hz, H3-18) 1.80-1.92 (m, H2-19) 5.23 (s, CH2 -O) 5.45 (s, H2-5) 5.57 (s, H2-17), 6.55 (s, -OH), 7.35 (s, H-14) 7.75-7, 95 (m, 2Ar; H-10; H-11), 8.2-8.4 (m, 2Ar; H-12), 8.65 (dd, J = 8.46Hz; J = 1.47 Hz, H-9), 9.50 (s, -CH = N).

The following compounds were obtained by the same procedure:

7-p-methylbenzyloxyiminomethylcamptothecin (CPT 178) mp. 203 ° C dec.

7-pentafluorobenzyloxyiminomethylcamptothecin (CPT 182 mp 200 ° C dec.

Claims (11)

1. Compounds of formula (I) wherein: R1 is the group -C (R5) = NO (n) R4 where R4 is straight or branched C1-C8-alkyl, or C6-C14-aryl selected from the group consisting of phenyl or benzyl, or heterocyclo-straight or branched C1- C8-alkyl wherein said heterocyclic group contains at least a heteroatom selected from the group consisting of a nitrogen atom optionally substituted with a (C1-C8) -alkyl group, and / or oxygen; wherein said heterocyclic group is selected from the group consisting of pyridyl, N-methylpiperidinyl, uracil, morpholinyl and oxiranyl; wherein the alkyl is optionally substituted with one or more groups selected from the group consisting of phenyl, -NR6R7 wherein R6 and R7 are hydrogen, or wherein said arylalkyl is optionally substituted with one or more groups selected from C1-alkyl, or halogen; or a pharmaceutically acceptable ester thereof; or R4 is a glycosyl group selected from 6-D-glucosyl, optionally substituted with a ketal group selected from the group consisting of 1,3-dioxolyl; n is 1; R5 is H; R2 is H; R3 is hydrogen, hydroxy, alkoxy wherein alkoxy is methoxy; single isomers thereof, and pharmaceutically acceptable salts thereof. 2. The compounds according to claim 1 1, which are: 7-methoxyiminomethylcamptothecin; 7-methoxyiminomethyl-10-hydroxycamptothecin; 7-t-butoxyiminomethylcamptothecin; 7-t-butoxyiminomethyl-10-hydroxycamptothecin; 7-t-butoxyiminomethyl-10-methoxycamptothecin; 7-triphenylmethoxyiminomethylcamptothecin; 7- (2-amino) ethoxyiminomethylcamptothecin; 7-benzyloxyiminomethylcamptothecin; 7-phenoxyiminomethylcamptothecin; 7-p-methylbenzyloxyiminomethylcamptothecin; 7-pentafluorobenzyloxyiminomethylcamptothecin; 7-p-phenylbenzyloxyiminomethylcamptothecin; 7-oxiranylmethoxyiminomethylcamptothecin; 7- [2- (1-uracil) ethoxy] iminomethylcamptothecin; 7- (4-pyridyl) methoxyiminomethylcamptothecin; 7 - [(N-methyl) -4-piperidinyl] methoxyiminomethyl camptothecin; 7 - [(2- (4-morpholininyl] ethoxy] iminomethylcamptothecin; and 7- (6-D-glucosyloxy) iminomethylcamptothecin. 3. The compound according to p. 2 which is 7- (t-butoxy) -iminomethylcamptothecin 4. The compound according to p. 2 which is 7-benzyloxyiminomethylcaptothecin. PL 222 208 B1 5. A process for the preparation of compounds of formula (I) wherein R4 is as defined above, excluding aroyl, comprising the reaction of a compound of formula (Ia) in which R1 is -C (R5) = O, wherein R5 is the meaning given for formula (I), R2 and R3 are as defined for formula (I), with a compound of formula (IIa) R ₄ O-NH ₂ , in which R ₄ is as defined above, obtaining compounds of formula (I) wherein R1 is -C (R5) = N-OR4, R4 is as defined for formula (I), with the exception of aroyl. 6. The method according to p. The compound of claim 5, wherein the ratio between the compound of formula (Ia) and the compound of formula (IIa) is between 1: 3 and 3: 1. 7. The method for the production of compounds according to the acc. Wherein R4 is as defined above, except aroyl, which comprises the reaction of a compound of formula (Ia) in which R1 is -CN-OH, where R5 is as defined in formula (I), R2 and R3 are as defined in formula (I), with a halide of formula R4-X, in which X is halogen and R4 is as defined above, compounds of formula (I) in which R1 is a group of formula -C (R5) = N-OR4, R4 is as defined for formula (I), with the exception of aroyl. Compounds according to any one of claims 1 to 8 1-4, as drugs. 9. A pharmaceutical composition comprising a therapeutically effective amount of a compound as defined in any one of claims 1 to 9. 1-4, in admixture with pharmaceutically acceptable vehicles and excipients, wherein the therapeutically effective amount ranges from 2 mg / kg to 25 mg / kg. 10. Use of a compound as defined in any one of claims 1 to 10 1-4, for the manufacture of a medicament useful in the treatment of tumors. 11. The use according to claim 1 10, for the manufacture of a medicament useful in the treatment of non-small cell lung cancer.